Measurement of local permeability at subcellular level in cell models of agonist- and ventilator-induced lung injury

• Published in: Laboratory investigation Vol. 93; no. 2; pp. 254 - 263
• Main Authors: Dubrovskyi, Oleksii, Birukova, Anna A, Birukov, Konstantin G
• Format: Journal Article
• Language: English
• Published: New York Elsevier Inc 01-02-2013; Nature Publishing Group US; Nature Publishing Group
• Subjects: 631/1647; 692/699/1785; Avidin - metabolism; Avidin - pharmacokinetics; Biological Transport - physiology; Biotinylation; Capillary Permeability - physiology; Cell Culture Techniques; Collagen - metabolism; Culture Media - chemistry; cytoskeleton; Endothelial Cells - metabolism; endothelial dysfunction; Fluorescein-5-isothiocyanate - analogs & derivatives; Fluorescein-5-isothiocyanate - metabolism; Fluorescein-5-isothiocyanate - pharmacokinetics; Fluorescent Antibody Technique; Gelatin - metabolism; Humans; intercellular gaps; Laboratory Medicine; Medicine; Medicine & Public Health; pathological mechanical strain; Pathology; permeability; Pulmonary Artery - cytology; research-article; Tissue Array Analysis - methods; vascular leak; Ventilator-Induced Lung Injury - physiopathology; visualization; intercellular gaps; visualization; cytoskeleton; pathological mechanical strain; permeability; vascular leak; endothelial dysfunction
• ISSN: 0023-6837; 1530-0307
• DOI: 10.1038/labinvest.2012.159

Alterations of cell monolayer integrity and increased vascular permeability are key to many pathologies, including atherosclerosis, stroke, lung injury, cancer, digestive disorders and others. Current approaches to probe cell permeability require specific culture conditions and provide an average estimation of trans-monolayer permeability, while analysis of regional monolayer permeability in static and mechanically challenged monolayer at a single-cell scale resolution remains unavailable. We describe a novel method for visualization and rapid quantification of trans-monolayer permeability based on high-affinity interactions between ligand (FITC-conjugated avidin) added in the culture medium, which permeates cell monolayer to reach substrate-bound acceptor (biotinylated gelatin or collagen). This approach was used to simultaneously evaluate general and local permeability responses by endothelial cell (EC) monolayer to a spectrum of barrier protective and barrier disruptive agonists and their combinations. The results revealed the paracellular pathway as the predominant mechanism of agonist-induced mass transport by pulmonary EC. We also detected for the first time, in a direct assay, a synergistic effect of pathologically relevant levels of cyclic stretch (CS) and edemagenic agent thrombin in the development of pulmonary EC hyper-permeability response observed in ventilator-induced lung injury. The reported novel assay provides unique information about local monolayer permeability changes induced by agonists, mechanical factors or molecular perturbations in single cells. However, the spectrum of substrates, assay formats and experimental conditions compatible with this assay suggest its broad application in the areas of endothelial and epithelial biology, cancer research and other fields.